Effect of PCR extension temperature on high-throughput sequencing

doi:10.1016/j.molbiopara.2010.11.013

Molecular and Biochemical Parasitology

Volume 176, Issue 1, March 2011, Pages 64-67

https://doi.org/10.1016/j.molbiopara.2010.11.013 Get rights and content

Abstract

The DNA amplification process can be a source of bias and artifacts, especially when amplifying genomic areas with extreme AT or GC content. The human malaria parasite Plasmodium falciparum has an AT-rich genome, and some of its highly AT-rich regions have been shown to be refractory to polymerase chain reaction (PCR) amplification. Biased amplification may lead to erroneous conclusions for studies investigating genome-wide gene expression, nucleosome position, and copy number variation. Here we compare genome-wide nucleosome coverage in libraries amplified at three different extension temperatures and show that reduction in PCR extension temperature from 70 °C to 60 °C can greatly increase the fraction of coverage at AT-rich regions of the P. falciparum genome. Our method will improve the efficiency and coverage in sequencing an AT-rich genome.

Graphical abstract

. Reduced PCR extension temperature increases high throughput sequencing coverage at AT-rich regions.

Research highlights

▶ Sequence coverages amplified at extension temperatures of 70 °C, 65 °C, and 60 °C were compared. ▶ Significantly increased sequence coverage at AT-rich regions when amplified at 60 °C. ▶ DNA with a wide range of AT content can be amplified reliably using an extension temperature of 60 °C.

Section snippets

Acknowledgments

We thank Artem Barski, Qingsong Tang, and Gang Wei for assistance with the Illumina sequencing. This work was supported by the Divisions of Intramural Research at the National Institute of Allergy and Infectious Diseases and National Heart, Lung and Blood Institute. We thank NIAID intramural editor Brenda Rae Marshall for assistance.

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Published by Elsevier B.V.

Short technical reportEffect of PCR extension temperature on high-throughput sequencing

Abstract

Graphical abstract

Research highlights

Section snippets

Acknowledgments

Cell

Trends Genet

Cell

Overview of DNA sequencing strategies

Curr Protoc Mol Biol

Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

BMC Genomics

Substantial biases in ultra-short read data sets from high-throughput DNA sequencing

Nucleic Acids Res

Impact of whole genome amplification on analysis of copy number variants

Nucleic Acids Res

Frt-seq: amplification-free, strand-specific transcriptome sequencing

Nat Methods

Single-molecule DNA sequencing of a viral genome

Science

Short technical report
Effect of PCR extension temperature on high-throughput sequencing